Small interfering RNAs (siRNA) in lipid complexes applied intravaginally induce durable gene silencing lasting for at least 9 days throughout the epithelium, lamina propria and stroma of mouse genital tissue, but not in distal organs. Topical application of siRNAs targeting herpes simplex virus type 2 (HSV-2) protects mice from lethal vaginal viral challenge. The goal of this project is to use mouse models to build on these promising preliminary data to develop an siRNA-based topical microbicide for HIV. This project will take advantage of the low cost and ease of manipulation of mouse models to examine the optimal siRNA formulation suitable for vaginal application and examine toxicity and pharmacokinetics at different stages of the menstrual cycle. Although the effectiveness of an siRNA-based microbicide to block HIV transmission cannot be tested in mice, the effect of varying siRNA formulations on inhibiting HSV-2 transmission will be evaluated. Since HSV-2 infection is an important cofactorfor HIV transmission, information about blocking HSV-2 in mice may be directly relevant to designing an ultimate formulation targeting both viruses. However, HSV-2 infects epithelial and neuronal cells, while HIV transmission is likely via Langerhans cells (LC) and lamina propria T cells, dendritic cells (DC) and macrophages. Therefore, HSV-2 protection does not necessarily translate into protection against HIV transmission. To achieve this goal, silencing must be achieved in the genital mucosa in the cells responsible for HIV transmission. This project will determine whether LC, DC, T cells and macrophages in the mouse genital tissue are silenced by siRNA-lipid complexes. If not, it will test in mice alternate cellular targeting strategies being developed in Project by Lieberman and the siRNA Manufacturing and Toxicology Core. Some of the questions to be answered include: How long do siRNAs reside in the vagina before uptake into cells? What is the duration of silencing in different cell types in situ? Does expression of the target gene affect the duration of silencing? How durable is protection from HSV-2 challenge after siRNA administration and how late after exposure can siRNAs be administered for post-exposure protection? How does menstrual variation affect uptake, gene silencing and protection in the HSV-2 model? Is there systemic exposure after topical administration? As siRNAs are optimized in the siRNA Manufacturing and Toxicology Core and Project by Lieberman as to sequence, chemical modification for stability and delivery, and formulation, their initial in vivo testing will be in this project. These results, with those from project by Lieberman, will be used to select lead candidates and design optimal primate experiments in Project by Veazey, where cost precludes testing too many variables.